The process of blood clotting typically begins with trauma to a blood vessel. The damaged vessel wall initiates hemostasis by causing adherence and accumulation of platelets at the injury site and by activating plasma proteins which initiate the coagulation process. A series of proteins, including Factor IX, are activated sequentially by specific proteolytic cleavages and conformational changes leading to the deposition of insoluble fibrin which curtails the blood flow.
Blood coagulation Factor IX circulates as an inactive zymogen. It enters the coagulation cascade after activation in the presence of Factor XIa or VIIa and tissue factor. The activated Factor IX (IXa) in turn activates Factor X in the presence of Factor VIIIa, phospholipids and calcium.
Hemophilia is a bleeding disorder caused by the lack of an essential blood factor. Hemophilia A (classic hemophilia) is the most common and is caused by a genetic deficiency or abnormality of Factor VIII. Hemophilia B is caused by a deficiency or abnormality of Factor IX. The genetic deficiency of Factor IX results in hemophilia B. It has been estimated that about 3000 patients in the U.S. alone suffer from hemophilia B.
Hemophilia is a heterogeneous disorder. Mutant blood Factor IX proteins can have clotting activities that vary from near normal to severely deficient. The clinical features of hemophilia A and B are identical. Less than 1% clotting factor activity is defined as severe hemophilia, and is accompanied by spontaneous bleeding in the muscles and larger joints. Repeated bleeding in joints causes arthropathy or hemarthroses which is a major chronic complication. Hemarthroses are worsened by muscle atrophy due to muscle bleedings. Often, repeated hemarthroses result in eventual deformity and crippling. In mild hemophilia (5-40% FVIII or FIX activity), bleeding does not occur except after trauma. Moderately severe hemophilia (1-5% FVIII or FIX activity) has clinical features between the severe and mild hemophilia.
Conventional treatment of hemophilia B consists of replacement of the deficient Factor IX from pooled donor plasma including fresh, frozen plasma or Factor IX concentrates. Pooled donor preparations, however, have been associated with the transmission of viral diseases such as hepatitis (eg. B, delta, non A non B) and HIV. This is in spite of increased purification techniques to reduce the virus load. It has been estimated that 50% of hemophilia patients are either hepatitis positive or HIV positive. These viral infections are now the major cause of morbidity and mortality in patients with hemophilia.
Further disadvantages of pooled donor plasma include the cost and availability of the purified blood factors. With increased purification steps, the cost of blood factor therapy has increased. Availability of the blood factors is also a concern. Theoretically, these factors should be administered prophylactically in many cases to avoid the sequelae of uncontrolled bleeding such as the development of joint disorders. However, cost, availability and the pharmacokinetics make an effective prophylactic therapy unfeasible.
With the advent of DNA technology, researchers have now cloned and are testing a number of recombinant blood factors, including Factor IX in patients with hemophilia. While recombinant technology may overcome the problems of viral contamination and availability, it does not affect the pharmacokinetics of the factors nor the formation of inhibitors (antibodies) in patients. It is estimated that 2-3% of all patients with hemophilia B will develop IgG antibodies that will nullify the value of replacement therapy. Inhibitor development occurs primarily in patients with severe hemophilia although antibodies to Factor IX in mild hemophilia have been reported. Approximately 5-15% of patients with severe hemophilia have antibodies to Factor VIII or IX. It has been estimated that the actual risk of developing neutralizing antibodies by age 20 is as high as 15-24%. Joint bleedings often cannot be controlled and adequately treated and many of these patients are severely handicapped.
To overcome the neutralizing effect of the antibodies, physicians can be forced to increase the dosage to the factor. However, there is often a decreased response to the replacement therapy despite increases in dosage. Care in administering the factors as well as the administration of steroids and other immunosuppressive agents such as azathioprine, cyclophosphamide and high-dose gammaglobulin G is often required to prevent or limit the development of antibodies and hypersensitivity reactions. Antibody depletion through plasmapheresis has been used to decrease the inhibitor titers in circulation. However, such immunosuppressive techniques have been only partially successful and raise the risk that the patient will be more vulnerable to opportunistic infections, e.g. HIV or hepatitis.
In light of the complications and risks inherent in the conventional treatment of hemophilia B, it is desirable to provide compositions having Factor IX activity which are less likely to cause the formation of inhibitor antibodies. In light of the high costs of Factor IX, it is also highly desirable to increase the in vivo circulating life of Factor IX activity.